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In vitro reconstituted human erythrocyte, its preparation and application in blood substitute material

A technology of human erythrocytes and erythrocytes, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of brittle membranes, inestimable demand markets, difficulties in storage and transportation of natural blood, and improve life expectancy Effect

Inactive Publication Date: 2004-08-11
FEIXIANG BIOTECH CHONGQING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are a series of problems in traditional personnel blood transfusion methods: 1. Natural blood storage and transportation are also very difficult: the storage time of blood is short, and the longest storage time is 42 days at 4°C-8°C, and it is easy to cause hemolysis if it is transported for a long distance
2. Due to the risk of various viruses including AIDS and hepatitis, although blood donors have undergone strict blood test screening, the risk of hepatitis C is still 1:3000, and the risk of HIV infection is 1:100000~1:1000000 ; Blood transfusions can also cause the transmission of other viruses, bacteria, parasites, and other diseases
3. Donor-recipient matching is required for blood transfusion, otherwise there will be immune reactions, compatibility of different blood types, lack of special blood types, etc.
4. Population aging, lack of healthy blood supply population, serious shortage of fresh blood in some areas, serious shortage of blood sources, etc., bring serious difficulties and challenges to clinical treatment and first aid; especially in wars, severe natural disasters, terrorist In emergencies, it is difficult to meet the actual demand only by blood supply from blood banks and personnel donations. Therefore, the demand market for blood substitutes, especially "reconstructed red blood cells" that is closest to normal red blood cells in terms of performance and function, is incalculable.
But its existing problems are: the oxygen-carrying efficiency of this kind of liposome artificial red blood cells is not high, the membrane is brittle, hemoglobin leaks, and the storage time is as short as ten minutes, and the longest is only a few days
The reason why the stability and oxygen-carrying function of liposome-encapsulated hemoglobin cannot meet the requirements is that previous studies have ignored the structural relationship between hemoglobin and structural proteins, and between structural proteins and membrane lipids, and the impact of this relationship on red blood cells. Contributes to excellent deformability and rheological properties while only physically encapsulating hemoglobin with membrane lipids

Method used

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  • In vitro reconstituted human erythrocyte, its preparation and application in blood substitute material
  • In vitro reconstituted human erythrocyte, its preparation and application in blood substitute material
  • In vitro reconstituted human erythrocyte, its preparation and application in blood substitute material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Separation and Purification Method I of Structural Protein

[0034] (1), extract structural protein

[0035] Take 5 grams of soybeans and soak them in 10-30ml of physiological saline for 8-12 hours, mash the tissue and filter to remove the residue; add 10-200 120mmol magnesium chloride saline solution to the emulsion, and freeze and centrifuge the solution (5000rpm when flocs are formed) , ~4°C) for 15-20 minutes, discard the supernatant, dissolve the precipitate with normal saline, then add ammonium sulfate solution to saturate the coagulated flocs, and then centrifuge (5000rpm, ~4°C) for 15-20 minutes , to obtain crude 11S globulin.

[0036] (2) Separation and purification of structural proteins

[0037] The above crude 11S globulin was desalted by dialysis (molecular weight cut-off 2 + : 10~200mmol) to make more than 70% of the product be αβ dimer globulin.

Embodiment 2

[0038] Example 2 Separation and Purification of Structural Protein II

[0039] (1), extract structural protein

[0040] Use blood containing anticoagulants (approximately 5ml of heparin pH7.4 isotonic phosphate buffer per 30ml of blood) in order to minimize the denaturation and enzymatic hydrolysis of structural proteins, the entire operation should be carried out at a low temperature of 0-4°C . Take 5ml of the above-mentioned anticoagulated blood, refrigerated and centrifuged (3000rpm, ~4°C, 15-30 minutes) to precipitate red blood cells, and separate the plasma supernatant and buffy coat.

[0041] The erythrocytes were washed 2 to 5 times with 3 times the volume of pre-cooled pH7.4 isotonic phosphate buffer, each time refrigerated and centrifuged (3000rpm, ~4°C, 15 minutes), and the supernatant and precipitated surface layer were removed.

[0042] Add pre-cooled 5-10 mmol / L, pH 7.4 hypotonic Tris (trishydroxymethylaminomethane)-HCl buffer solution at a volume ratio of 10-50...

Embodiment 3

[0045] Embodiment 3 The ultraviolet absorption spectrum of structural protein

[0046] The structural protein product obtained by separation and purification is dissolved in physiological saline at a concentration of 200-800 ppm, and subjected to ultraviolet spectrophotometric analysis. Instrument: PE-λ900, scanning wavelength range: 200-400nm. For spectrogram see figure 2 . The solid lines in the figure represent the ultraviolet absorption peaks of membrane proteins derived from animal red blood cells, and the dotted lines represent the ultraviolet absorption peaks of 11S globulin derived from plant proteins. The three spectral curves all show the maximum absorption peak at 280nm.

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Abstract

A human red cell recombined in vitro, its preparing process and it application to blood substitute are disclosed. The said human red cell is composed of haematoglobin, structural protein and phospholipid and has the physiological function and form, which are similar to that of natural red cell. Its advantages are good deformability, good stability, strong oxygen carrying and releasing power, and controllable size.

Description

technical field [0001] The invention relates to an artificial erythrocyte reconstructed in vitro by structural protein (membrane protein of erythrocyte or soybean 11S globulin), phospholipid, hemoglobin, etc., as a new blood substitute. The present invention uses structural protein and phospholipid as the basic molecular materials to synergistically encapsulate hemoglobin to reconstruct artificial red blood cells in vitro; through the combination of hemoglobin, structural protein and phospholipid, the physiological function and shape of the reconstructed in vitro are close to natural red blood cells, with deformability and stability Good, strong oxygen-carrying-oxygen-releasing capacity, deimmunized, and the cell size can be controlled to the nanometer scale to rebuild red blood cells. The invention relates to an artificial red blood cell reconstructed in vitro by structural protein, phospholipid, hemoglobin, etc., as a new blood substitute. Background technique: [0002] S...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078
Inventor 王翔段建军高玮
Owner FEIXIANG BIOTECH CHONGQING
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